1. Field of the Invention
This invention relates to lysing agents, more particularly, lysing agents for analysis of hemoglobin.
2. Discussion of the Art
The science of hematology has long recognized the importance of measuring the amount of hemoglobin in a blood sample, because it is the hemoglobin molecule that transports oxygen from the lungs to the various tissues and organs of the body. It can be argued that the accurate measurement of the concentration of hemoglobin in a patient's blood is the most important parameter in a hematology analysis. The concentration of hemoglobin is used to screen for anemia, which, in turn, is a sign of underlying disease.
A concentration of hemoglobin below 14 grams per deciliter (g/dL) in men and 12 g/dL in women is indicative of anemia. The causes of anemia are many, and a low concentration of hemoglobin is a strong signal for a thorough workup by a patient's physician. The two most common reasons for a patient to be anemic are blood loss and dietary deficiencies in iron, vitamin B12, or folic acid. In the case of anemia, it is mandatory for the patient's physician to determine the cause of loss of blood and treat it. In the case of a vitamin deficiency or a mineral deficiency, a proper diagnosis is needed to determine the appropriate nutritional supplement(s).
In addition to its importance as the primary indicator of anemia, the concentration of hemoglobin is used in combination with other parameters of blood cells to calculate several indices. The value of mean corpuscular hemoglobin (MCH), which is the mass of hemoglobin per red cell, is calculated by dividing the concentration of hemoglobin by the concentration of red blood cells. The mean corpuscular hemoglobin concentration (MCHC) is the weight percent of hemoglobin in a red blood cell and is calculated by dividing the concentration of hemoglobin by the hematocrit and converting the quotient to a percent. Both MCH and MCHC are useful parameters in the diagnosis of anemia.
Modern methods of measuring the concentration of hemoglobin utilize spectrophotometry to quantify the amount of the oxygen-carrying protein in a sample of blood. The requirements of any spectrophotometric method are two-fold:                (1) The method must release all the hemoglobin from the red blood cell in which it is sequestered.        (2) The method must convert all the hemoglobin in the sample into a single chromogenic species, regardless of the form in which the hemoglobin existed when the binding reaction between the ligand and protein began.        
The first requirement can be achieved by diluting a sample of blood in distilled water to effect a hypotonic lysis. However, modern automated hematology analyzers require a more rapid lysis than can be achieved with hypotonic lysis. Frequently, surfactants are added to the lysing agent to hasten the release of hemoglobin and to clear any turbidity. Various classes of surfactants are suitable for this task, including anionic, non-ionic, zwitterionic, and cationic. The amount of surfactant required can range from about 100 mg/L to about 50 g/L, depending on the potency of the surfactant and other features of the lysing agent, such as, for example, pH, osmolality.
The second requirement necessitates an understanding of the chemistry of the heme iron, which carries oxygen when complexed in a globin protein molecule. The heme iron is maintained in the Fe+2 oxidation state in a sample of normal blood. Because a sample of blood is usually drawn from a vein, the hemoglobin is usually in the deoxy state, i.e., no oxygen is bound to the heme iron. However, as soon as the sample comes into contact with the ambient air, or is introduced into an oxygen-containing buffer or lysing agent, the hemoglobin is rapidly converted into oxy-hemoglobin; the heme iron binds oxygen but stays in the Fe+2 (reduced) state. In many cases, the amount of hemoglobin in the sample can be determined from the oxy-hemoglobin chromogen, which is formed naturally upon exposure of the sample to air. However, in some diseases, genetic conditions, or poisonings, a patient may have a significant amount of methemoglobin in circulation. In methemoglobin, the heme iron is in the Fe+3 oxidized state. The heme iron cannot bind oxygen, nor can it be readily reduced to Fe+2 so that the heme iron can bind oxygen to be measured as oxy-hemoglobin. Heavy cigarette smokers and workers exposed to high concentrations of automobile exhaust frequently accumulate a high concentration of carbon monoxide bound to their heme iron. Carbon monoxide is tightly bound and blocks the binding of oxygen, thereby causing an error in the measurement of the concentration of hemoglobin if the concentration of hemoglobin is determined by the oxy-hemoglobin method. The most commonly used approach for the measurement of hemoglobin is to oxidize all of the heme iron to the Fe+3 oxidized state and to introduce a ligand that will quantitatively bind to all the heme iron in the Fe+3 oxidized state to produce a single chromogenic species for quantification by spectrophotometry.
The classical method for measuring the concentration of hemoglobin is that of Drabkin. The hemoglobin is released from the sample of blood by hypotonic lysis, the heme iron is oxidized to the Fe+3 oxidized state by means of potassium ferricyanide [K3Fe(CN)6], and the iron is reacted with the cyanide anion of the potassium cyanide (KCN). Cyanide binds very tightly to Fe+3 and gives a distinctive chromogen with a peak at a wavelength of about 540 nanometers (nm).
Traditional methods for analyzing hemoglobin employ (1) at least one quaternary ammonium salt as a lysing agent to destroy erythrocytes and (2) potassium cyanide (KCN) as a binding ligand to bring about rapid conversion of hemoglobin to the cyanomet-derivative of hemoglobin. The rapid binding of cyanide to hemoglobin and the extremely stable absorption spectrum of cyanmethemoglobin (peak ε=12.5 mM−1 cm−1 at a wavelength of about 540 nm) ensure accurate count of hemoglobin in a sample of whole blood and in a sample of control/calibrator.
However, the use of cyanide in a lysing agent raises safety and environmental concerns, and the handling and disposal of waste material is costly and presents risks to the environment. Yet, the cyanmethemoglobin method for analyzing hemoglobin is still deemed the most preferred method for analyzing hemoglobin.
Accordingly, a great deal of effort has been expended over the past twenty years to develop methods for analyzing hemoglobin without the need for using cyanide. The major suppliers in the hematology industry have adopted cyanide-free methods for analyzing hemoglobin in their state-of-the-art hematology analyzers. These cyanide-free methods for analyzing hemoglobin require using one or more binding ligands for hemoglobin in order to achieve equivalent absorbance at the typical measurement wavelength (540 to 560 nm). Cyanide-free methods have eliminated the cyanide reagent. See, for example, U.S. Pat. Nos. 5,631,165; 5,939,326; 5,612,223; 5,866,428; 5,958,781; 6,740,527; 6,890,756, all of which are incorporated herein by reference.
The methods described in U.S. Pat. Nos. 5,612,223 and 5,866,428 use a cyanide-free reagent comprising an aqueous solution of (i) a cyanide-free ligand selected from the group consisting of imidazole, imidazole derivatives, N-hydroxyacetamide, N-hydroxylamine, pyridine, oxazole, thiazole, pyrazole, pyrimidine, purine, quinoline, and isoquinoline and (ii) a strong erythrolytic surfactant selected from the group consisting of lauryl dimethylamine oxide and octylphenoxy ethanol. The pH of the reagent is adjusted to from about 11 to about 14, preferably with a monovalent base. According to the methods, the cyanide-free reagent is rapidly mixed with the blood sample to form a chromogen. The absorbance, or optical density, of the resulting chromogen is then measured to provide an indication of the concentration of hemoglobin. The methods described in U.S. Pat. Nos. 5,958,781 and 6,740,527 use a cyanide-free reagent comprising (1) at least one quaternary ammonium salt selected from the group consisting of tetradecyltrimethyl ammonium bromide, dodecyltrimethyl ammonium chloride, cetyl trimethyl ammonium bromide, hexadecyltrimethyl ammonium bromide, benzalkonium chloride, and cetyl pyridium chloride and (2) at least one hydroxylamine salt selected from the group consisting of hydrochloride, sulfate, phosphate, and other acid salts. A chromogen is formed, detected, and measured, thereby indicating concentration of hemoglobin in a sample of whole blood as well as white blood cell population and subpopulation determinations. The method described in U.S. Pat. No. 6,890,756 uses a cyanide-free lyse solution including a quaternary ammonium salt surfactant, an anionic surfactant, a hemoglobin binding agent selected from the group consisting of imidazole and hydroxylamine, and an aqueous medium.
However, the foregoing cyanide-free methods require the use of ligands for binding hemoglobin. These binding ligands are undesirable because of safety, health, and cost considerations. Accordingly, it would be desirable to develop simplified lysing agent formulations without the use of binding ligands.